We utilized a sensitive, quantitative assay for iron regulatory protein 2 which allows study of endogenous iron regulatory protein 2 degradation in HEK293A cells under more physiologic conditions. We found that under these conditions the proteasome plays only a minor role in the degradation of iron regulatory protein 2, with almost all the iron regulatory protein 2 being degraded by a non-proteasomal pathway. This new pathway is calcium-dependent but is not mediated by calpain. Elevating the cellular level of iron regulatory protein 2 by inducing iron deficiency or by transfection causes the proteasomal pathway to account for the major fraction of iron regulatory protein 2 degradation. We conclude that under physiological, iron-sufficient conditions, the steady state level of iron regulatory protein 2 in HEK293A cells is regulated by the non-proteasomal pathway. We then performed DNA microarray analysis in an attempt to detect mRNA of protease(s) which were up-regulated when the non-proteasomal system was activated. We used the Affymetrix U133 human genome array which interrogates 33,000 human genes, but no upregulated protease genes were detected. We then tested chemical inhibitors of various classes of proteases for their ability to block the non-proteasomal degradation of iron regulatory protein 2. The only compound which did blunt the degradation was PD150606, a calpain inhibitor. However, detailed investigations established that calpain is not the protease responsible for the non-proteasomal degradation of iron regulatory protein 2. We have not yet identified the responsible protease.